Microfabricated apparatus for cell based assays

ABSTRACT

Apparatus and methods are provided for performing cell growth and cell based assays in a liquid medium. The apparatus comprises a base plate supporting a plurality of micro-channel elements, each micro-channel element comprising a cell growth chamber, an inlet channel and an outlet channel, a cover plate positioned over the base plate to define the chambers and connecting channels. Means are incorporated in the cell growth chambers, for cell attachment and cell growth. In particular, the invention provides a rotatable disc microfabricated for performing cell growth and cell based assays. The apparatus and method can be used for the growth of cells and the detection and measurement of a variety of biochemical processes and products using non-invasive techniques, that is techniques which do not compromise the integrity or viability of cells.

[0001] The present invention relates to cell based assays. In particularthe invention relates to a microfabricated apparatus for performing cellgrowth and cell based assays and to methods for performing such assays.

[0002] The current focus in high throughput screening applicationstowards the screening of increasing numbers of compounds is being drivenby the twin technologies of combinatorial chemistry and genomics, inorder to produce new potential drug targets and novel candidate drugs aspotential therapeutic compounds. The primary screening process has beenaddressed by the development of high throughput screening assayprocesses and assay miniaturisation utilising the microtitre well plateformat with 384, 864, 1536 or greater miniaturised wells. Miniaturisedassays are capable of allowing throughput levels of over 100,000tests/day in primary screening. At this level of throughput, a primaryscreen might be expected to yield 100-1000 ‘hits’ per day. Each of theseputative drugs is required to undergo further refined screening andtesting in a variety of assays in order to investigate the biologicalcompatibility of the compound. Such assays include bioavailability,metabolism and toxicology, and they are carried out predominantly usingcultured cell lines. In comparison with assays used in the primaryscreening process, secondary. screening assays have a much higher levelof complexity and more stringent requirements, both in the mechanics ofthe assay and in the information generated. There is a requirement inthe art for secondary screening assay methodologies which are capable ofhandling both the increasing rate of putative drug lead generation andthe generation of biological data concerning the drug candidate. Inaddition, development of assays yielding higher information content havethe potential to increase the level of characterisation of a lead drugduring the screening phase of drug development.

[0003] Microfabricated devices have been described previously which aresuitable for use in miniaturized biological analyses. For example, WO96/15450 discloses a device which comprises an etched glass structurewith a collection of chambers connected by a micro-channel and enclosedby a glass cover plate. Devices have been described by Wilding et al.,for example in WO 93/22058, which discloses a mesoscale device foramplifying DNA by PCR, the device consisting of a number of chambersconnected by a channel. WO 93/22055 and WO 93/22053 relate to devicesfor analysing a fluid cell-containing sample comprising a mesoscale flowsystem with entry port for capture and/or lysis of cells in biologicalfluids, or containing a binding moiety for specifically binding ananalyte, where the analyte may be an intracellular component in a cellin a sample, or a cell population in a sample. The above devices areconcerned with the measurement or detection of cells or cellularanalytes in cells introduced into the device immediately prior toanalysis. They are not described for use in cell culture or growth, norfor use in the study of cellular responses to agents under test.Moreover, they do not describe or permit the use of adherent culturedcells.

[0004] There is a requirement for a device capable of providing anenvironment which supports the long term survival of cultured cells,coupled with means to utilise cells cultured within the device forsecondary drug screening or other studies.

[0005] In one aspect, the present invention provides apparatusmicrofabricated for performing cell growth and cell based assays in aliquid medium, said apparatus comprising:

[0006] a) a base plate supporting a plurality of micro-channel elements,each comprising a cell growth chamber, an inlet channel for supplyingliquid sample thereto and an outlet channel for removal of liquid sampletherefrom;

[0007] b) a cover plate positioned over said base plate said cover plateextending over said elements so as to define said chambers andconnecting channels; said cover plate being supplied with holes toprovide access to said channels; and

[0008] c) means, incorporated in said cell growth chambers, for cellattachment and cell growth.

[0009] In a second aspect of the invention there is provided a methodfor studying the effect of a test substance on a cellular activity orphysical parameter by the use of the apparatus as defined, which methodcomprises:

[0010] a) providing a suspension of cells in a fluid medium;

[0011] b) introducing said cells into said apparatus and causing saidcells to be transported to one or more cell growth chambers in saidapparatus;

[0012] c) providing one or more samples of test substances whose effectupon the cells is to be measured under conditions so as to cause saidcells to be exposed to said substances;

[0013] d) determining the effect of the test substances on said cells bymeans of optical detection.

[0014] Preferably the method for studying the effect of a test substanceincludes the step of culturing cells adhering to a surface within theapparatus prior to the introduction of the test substances. Preferablythere are provided following step c) one or more assay reagents anddispersing the reagents to one or more reaction chambers in theapparatus.

[0015] In a further aspect of the present invention there is provided amethod for measuring a cellular analyte by the use of the apparatus asdefined, which method comprises:

[0016] a) providing a suspension of cells containing an analyte to bemeasured in a fluid medium;

[0017] b) introducing cells into the apparatus and causing the cells tobe transported to one or more cell growth chambers in the apparatus;

[0018] c) allowing cells to grow;

[0019] d) providing one or more assay reagents and dispersing thereagents to one or more chambers in the apparatus;

[0020] e) measuring the cellular analyte by optical means.

[0021] In order that the invention may be better understood, severalembodiments will now be described by way of example only and withreference to the accompanying drawings in which:

[0022]FIG. 1a is a diagrammatic representation in plan of an individualmicro-channel element of the microfabricated apparatus for performingcell growth and cell based assays;

[0023]FIG. 1b is a sectional view of a microfabricated disc including aplurality of assay elements for performing cell growth and cell basedassays according to the present invention;

[0024]FIG. 2 represents an alternative configuration of an individualassay element of the microfabricated apparatus for use with cellsgrowing on the surface of microcarrier beads; and

[0025]FIG. 3 represents a further configuration of an individual assayelement of the microfabricated apparatus in which means are provided forpreventing or impeding the passage of cells in the cell growth chamber.

[0026] Referring to FIG. 1b, the apparatus of the present inventioncomprises a rotatable disc (18) microfabricated to provide a sampleintroduction port (not shown) located towards the centre of the disc andconnected to an annular sample reservoir (9) which in turn is connectedto a plurality of radially dispersed micro-channel assay elements (6)each of said micro-channel elements comprising a cell growth chamber, asample inlet channel and an outlet channel for removal of liquidtherefrom and a cover plate positioned onto said disc so as to defineclosed chambers and connecting channels. Each micro-channel element isconnected at one end to the central sample reservoir (9) and at theopposing end to a common waste channel (10).

[0027] Each of the radially-dispersed micro-channel elements (6) of themicrofabricated apparatus (shown in FIG. 1a) comprises a sample inletchannel (1) connected at its left hand-end end to the reservoir (9), acell growth chamber (2) for performing cell growth and connected througha channel (4) to an assay chamber (3) and an outlet channel (5)connected at its right-hand end to the waste channel (10).

[0028] In an alternative format of the present invention as shown inFIG. 2, the micro-channel elements (6) are modified to permit the use ofthe apparatus with cells growing on the surface of microcarrier beads(17). Thus, each micro-channel element (6) comprises a sample inletchannel (8) connected at its left-hand end to the sample reservoir (9),a cell growth chamber (7) connecting through a channel (16) to an assaychamber (11), and an outlet channel (12) leading to the common wastechannel (10).

[0029] With reference to FIG. 3, in a further embodiment of theapparatus, the cell growth chamber (2) may be provided with raisedmoulded structures disposed on the base portion of the cell growthchamber to form pillars (15), such that they form a barrier to the flowor passage of cells arriving in the cell growth chamber (2) through theinlet channel (1), while allowing the passage of liquid. Thesestructures are of dimensions chosen to provide gaps between thestructures which are too narrow to allow passage of cells carried as asuspension in liquid moving in the device, but which are sufficientlylarge to allow cells growing on the surface of the cell growth chamber(14) to migrate through the barrier by extension of cell processesbetween the barrier and subsequent cytokinesis. Suitable dimensions forthe gaps between the raised pillars (15) formed in the cell. growthchambers are between 5 and 50 μm, depending upon the cell type and cellsize selected for capture.

[0030] Preferably, each micro-channel element (6) shown in FIG. 3additionally includes one or more assay chambers for performing assaysinvolving cellular constituents and being connected in line between saidcell growth chamber (2) and said outlet channel (5).

[0031] Suitably the disc (18) is of a one- or two-piece mouldedconstruction and is formed of an optionally transparent plastic orpolymeric material by means of separate mouldings which are assembledtogether to provid a closed structure with openings at defined positionsto allow loading of the device with liquids and removal of wasteliquids. In the simplest form, the device is produced as twocomplementary parts, one or each carrying moulded structures which, whenaffixed together, form a series of interconnected micro-channel elementswithin the body of a solid disc. Alternatively the micro-channelelements may be formed by micro-machining methods in which themicro-channels and chambers forming the micro-channel elements aremicro-machined into the surface of a disc, and a cover plate, forexample a plastic film, is adhered to the surface so as to enclose thechannels and chambers.

[0032] To provide cultured cells with means for obtaining oxygen formetabolism and to permit use of CO₂-buffered media, one or morecomponents of the device may be constructed from a gas permeableplastic, film, polymer, or membrane. Suitably, the gas permeableplastic, film, polymer, or membrane is formed from silicone polymers,such as polydimethylsiloxane, or from polyurethane,polytetrafluoroethylene or other gas permeable plastic materials. Inaddition, means (not shown) are preferably provided for sealing theopenings in the closed structure to prevent evaporation of liquid duringuse, whereby such means seal the openings without interfering with gasexchange to the cell growth medium. Sealing can be accomplished by useof a further layer of gas permeable material across the whole device, orby use of a non-permeable material applied locally to the openings only,leaving the remainder of the gas permeable material exposed to the localatmosphere.

[0033] Suitable plastic or polymeric materials for forming the cellgrowth chamber and micro-channels are preferably selected to havehydrophobic properties, where the surface of the plastic or polymer canbe additionally selectively modified by chemical or physical means toalter the surface properties to confer a desired property, for example,compatibility with cell growth, cell attachment and the attachment ofbiomolecules by covalent or non-covalent means. Preferred plastics areselected from polystyrene and polycarbonate.

[0034] Alternatively, the cell growth chamber and micro-channels may beconstructed from plastic or polymeric materials which are selected tohave hydrophilic properties. The surface of the plastic or polymer canbe additionally selectively modified by chemical or physical means toalter the surface properties so as to produce localised regions ofhydrophobicity within the chambers and/or microchannels to confer adesired property. By this means, for example, hydrophobic barriers orvalves may be provided to control liquid flow within the apparatus.Preferred plastics are selected from polymers with a charged surface,suitably chemically or ion-plasma treated polystyrene, polycarbonate orother rigid transparent polymers.

[0035] The micro-channel elements (6) are dispersed radially around thedisc (18) and connected to a common centre port. The channels 1, 4, 5,8, 12, and 16 are of a dimension compatible with movement of cells alongthe channels. Suitably, the channels and chambers may be of anycross-sectional shape, such as square, rectangular, circular, trapezoidand triangular. Suitably, the cell growth chamber (2) is sized to give afloor area between 100 μm² and 1,000,000 μm², preferably between 1000μm² and 1,000,000 μm² and most preferably between 10,000 μm² and1,000,000 μm². The plastic or polymer surface of the cell growth chambermay be selectively treated or modified to permit cell attachment and/orgrowth. Treatment preferably involves exposure to intense UV light tomodify the polymer surface or alternatively the use of high voltageplasma discharge using known techniques (see Amstein, C. F. andHartmann, P. A., J.Clin.Microbiol., 2,1, 46-54 (1975)) to create anegatively charged surface suitable for cell growth and attachment and(if required) for assay purposes. Cell attachment may be furtherimproved by the application of additional coatings to the surface of thecell growth chamber, eg. polylysine, collagen, fibronectin.

[0036] As already mentioned, in the preferred aspect of the invention,each of the micro-channel elements (6) is further provided with an assaychamber (3) which is located in line between the cell growth chamber (2)and the outlet channel (5), for performing assays involving cellularconstituents. The assay chamber is sized proportionally to the volume ofthe cell growth chamber (2) to allow collection and/or capture ofsoluble analytes which may be derived from the cultured cells understudy. Suitably, the volume of the assay chamber (3) is between twiceand one tenth of the volume of the cell growth chamber (2).Advantageously, the channel (4) which connects the cell growth chamberand the assay chamber is characterised by having hydrophobic walls and across-sectional area which is smaller than the channel (1) upstream ofthe cell growth chamber. Suitably, channel (4) has a cross-sectionalarea of between 0.99 and 0.01 times that of the inlet channel (1).Suitably, the outlet channel (5) is characterised by having hydrophobicwalls and a cross-sectional area of between 0.99 and 0.01 times that ofchannel (4). In this way, liquid flow in the micro-channel elements canbe controlled by application of a defined centrifugal force to cause theliquid to flow in a channel of a defined cross-sectional area, whereinthe same force is insufficient to cause the liquid to flow in furtherlinked channels of lesser cross-sectional area, this having the effectof stopping the liquid flow at a desired position in the micro-channelelement.

[0037] In an alternative means of controlling liquid flow, channel (1)and channel (4) are constructed with the same cross-sectional area, butthe surface of each of the channels may be selectively modified bychemical or physical means to confer a different degree ofhydrophobicity on that channel. For example, channel (4) downstream ofthe cell growth chamber (2) may be treated to have a higherhydrophobicity than the channel (1) upstream of the cell growth chamber.By this means, application of a defined force to liquid in channel (1),sufficient to cause liquid to move down this channel will beinsufficient to cause that liquid to enter the second channel (4) ofhigher hydrophobicity, this having the effect of stopping the liquidflow at a desired position in the micro-channel element. Suitable meansfor selectively modifying the hydrophobicity of the surface of thechannels include exposure of defined areas to ionizing orelectromagnetic radiation or ion plasma through a mask, or byapplication of a hydrophobic material or ink by screen printing or othermeans of localized application.

[0038] The means of controlling liquid flow within the apparatus asdescribed above may be used alone or in combination as required, inorder to confer the desired control of liquid flow in connected chambersand channels within the micro-channel element. If used in combination,the methods may be used sequentially, for example where a change incross-sectional area is followed by a change in hydrophobicity, formingtwo sequential points of liquid flow control. Alternatively, the methodsmay be used coincidentally, in which a change in cross-sectional area ofa channel is accompanied by a change in hydrophobicity of that channel,the combination being used to confer a degree of control over liquidflow not attainable using a single means.

[0039] The inner surface of the assay chamber may be coated with one ormore ligands capable of specifically binding an analyte of interest bycovalently or non-covalently linking the ligand to the surface. Examplesof ligands suitable for the purpose include: biotin, streptavidin,protein A, antibodies, lectins, hormone receptors, nucleic acid probes,DNA binding proteins and the like.

[0040] The apparatus and method can be used for the growth of cells andthe detection and measurement of cellular activity, cellular parametersand biochemical processes, for example cellular metabolism, cellviability, reporter gene expression, using non-invasive techniques, thatis techniques which do not compromise the integrity or viability ofcells. Alternatively, the apparatus may be used in the detection andmeasurement of cell-derived products, which have been released orotherwise excreted from cells, such as peptide hormones, secondmessengers, etc. By use of the apparatus shown in FIG. 3, the apparatuspermits studies of cell migration in response to chemical or physicalstimuli, for example chemotaxis, wherein a chemoattractant substance isplaced on one side of a barrier and cells placed on the opposite side ofthe barrier are observed to penetrate the barrier in moving toward thechemoattractant.

[0041] The invention may be used with any cell type that can be culturedon standard tissue culture plastic-ware. Such cell types include allnormal and transformed cells derived from any recognised source withrespect to species (eg. human, rodent, simian), tissue source (eg.brain, liver, lung, heart, kidney skin, muscle) and cell type leg.epithelial, endothelial). In addition, cells which have been transfectedwith recombinant genes may also be cultured using the invention. Thereare established protocols available for the culture of diverse celltypes. (See for example, Freshney, R. I., Culture of Animal Cells: AManual of Basic Technique, 2^(nd) Edition, Alan R. Liss inc. 1987). Suchprotocols may require the use of specialised coatings and selectivemedia to enable cell growth and the expression of specialist cellularfunctions. None of such protocols is precluded from use with theapparatus of this invention.

[0042] The scale of the device will to a certain extent be dictated byits use, that is the device will be of a size which is compatible withuse with eukaryotic cells. This will impose a lower limit on any channeldesigned to allow movement of cells and will determine the size of cellcontainment or growth areas according to the number of cells present ineach assay. An average mammalian cell growing as an adherent culture hasan area of ˜300 μm²; non-adherent cells and non-attached adherent cellshave a spherical diameter of ˜10 μm. Consequently channels for movementof cells within the device are likely to have dimensions of the order of20-30 μm or greater. Sizes of cell holding areas will depend on thenumber of cells required to carry out an assay (the number beingdetermined both by sensitivity and statistical requirements). It isenvisaged that a typical assay would require a minimum of 500-1000 cellswhich for adherent cells would require structures of 150,000-300,000μm², i.e. circular ‘wells’ of ˜400-600 μm diameter.

[0043] The configuration of the micro-channels in the present inventionis preferably chosen to allow simultaneous seeding of the cell growthchamber by application of a suspension of cells in a fluid medium to thesample reservoir by means of the sample inlet port, followed by rotationof the disc (18) by suitable means at a speed sufficient to causemovement of the cell suspension outward towards the periphery of thedisc by centrifugal force. The movement of liquid distributes the cellsuspension along each of the inlet micro-channels (1, 8) towards thecell growth chambers (2, 7). The rotation speed of the disc is chosenprovide sufficient centrifugal force to allow liquid to flow to fill thecell growth chamber (2, 7), but with insufficient force for liquid toenter the restricted channel (4, 16) of smaller diameter on the opposingside of the cell growth chamber.

[0044] Once rotation has stopped, cells in the suspension can settleunder gravity onto the bottom of the cell growth chamber (2, 7) andattach to the treated surface if present. Cells which remain in thechannel are unable to attach to the untreated hydrophobic surface andwill remain in suspension. Therefore, following an appropriate period oftime, chosen to allow cell attachment to take place, the device may berotated at a higher speed than previously, such that all liquid andunattached cells is caused to move towards the periphery of the discinto the waste channel (10) disposed around the edge of disc. Followingclearance of channels of unattached cells, rotation is slowed to thesame speed initially used to fill the micro-channel elements and freshcell culture media is applied to the sample reservoir. By rotation ofthe disc once more, the cell culture media flows under centrifugal forceto fill the micro-channels and the cell growth chambers as previouslydescribed. This action leaves the attached cells seeded on the surfaceof the cell growth chambers covered in fresh culture media suitable forcell growth and for cell based assays.

[0045] In the apparatus of FIG. 2, the beads (17) provide a largesurface area for cell attachment and growth in a relatively small volumeof liquid. In the device described herein, the beads (17) perform twofunctions; firstly they provide a surface for the attachment and growthof cells, so removing the need to provide a cell-growth compatiblesurface within the disc allowing a wider range of materials to be usedfor construction, and secondly they provide a larger physical carrierfor cells which allows channels within the device to be easily designedto prevent access of cells. In the preferred embodiment, each elementcomprises a cell growth chamber (2, 7), an analysis chamber (3, 11), anda waste chamber (10) which are arranged radially about the centre of thedisc and connected to a common port in the centre of the disc.

[0046] Following the addition of cell samples to the apparatus, theirattachment and subsequent cell growth, reagents and samples for assaymay be introduced into the sample reservoir (9) through the sample inletport. Reagents to be supplied to all micro-channel elements may beintroduced as described hereinbefore for seeding with cells, that is byapplying a solution containing reagents to the central port and rotatingthe disc so as to cause distribution of the reagent to allmicro-channels, cell growth chambers and assay chambers. The applicationof specific samples to specific wells may be achieved by pipetting smallvolumes of liquid as discrete drops into the central well directlyadjacent to the opening of the inlet channel leading to the cell growthchamber which is to receive the sample. This may be achieved, forexample, by the use of a piezo-electric dispensing device (not shown),whereby dispensing of droplets of liquid from the device is synchronisedwith the speed of rotation of the disc (18) such that, at an appropriatecombination of dispensing drop frequency and rotation speed of the disc,individual droplets of liquid can be caused to impinge on the surface ofthe disc and can be caused to be transported by centrifugal force intoan adjacent inlet channel (1, 8) and to mix with liquid present in thecell growth chamber (2, 7).

[0047] Alternatively liquids may be pipetted as discrete drops onto thehydrophobic surface of a stationary disc (18), rotation of the discbeing used to move these drops of liquid into the appropriate inletchannel and so to the cell growth chamber. By these means, all reagentsand samples may be provided to the cell chamber in the desired order andproportions to perform the assay.

[0048] Once the liquid manipulations of the assay have been performed itis necessary to perform a detection procedure to measure the result ofthe assay, typically by measuring the signal emitted by a fluorescent,chemiluminescent or other labelling moiety. The device as shown in FIG.1b is configured to allow two means of detection of the assay signal.Firstly, the assay signal may be measured in-situ in the cell growthchamber. Such a signal would be typified by the measurement of theproduct of a reporter gene within the cells, e.g. fluorescence from GFP.

[0049] Alternatively, it may be desirable to measure cell-derivedproducts or analytes, for example by immunochemical or other specificbinding assay methods, in the absence of cells cultured in theapparatus, so as to avoid interference in measurement. In this case, theapparatus is provided with a second chamber, ie. the assay chamber (3,11) disposed closer to the periphery of the disc and connected to thecell growth chamber (2, 7). The assay chamber is connected to theperipheral waste channel (10) by a narrow channel (5, 12), this channelhaving a smaller diameter than that (4, 16) connecting the assay chamberto the cell growth chamber. The differential in diameters betweenchambers allows, under controlled conditions of rotation and centrifugalforce, as discussed above, liquid to be moved from the cell growthchamber to the assay chamber. For example, by this process, an analyteis moved from the cell growth chamber into the assay chamber where theanalyte is subject to affinity capture by a ligand attached to the wallof the assay chamber. This process therefore provides a means of movinga cell-derived analyte, which has been released or otherwise excretedfrom cells, away from the cellular environment, immobilising the analytewithin the assay chamber by means of a specific binding partner forbinding to the analyte, and allowing subsequent additions of reagentsand processing to permit the detection of the analyte.

[0050] Optionally, at least one of the reagents for use in an assaymethod utilising the apparatus may be labelled with a detectable labelby covalent or non-covalent attachment. Suitable detectable labels maybe selected from fluorescent labels, chemi-luminescent labels,bio-luminescent labels, enzyme labels and radioactive labels. Suitablefluorescent labels for tagging reagents according to the method of theinvention may be selected from the general categories of fluorescentdyes, including, but not limited to, fluoresceins, rhodamines, cyaninedyes, coumarins, and the BODIPY groups of fluorescent dyes. Examples ofbioluminescent detectable labels are to be found in the fluorescentreporter proteins, such a Green Fluorescent Protein (GFP) and aequorin.Alternative labels for providing a detectable signal can be fluorescenceenergy transfer labels.

[0051] Once the assay has been completed, measurement of the signal maybe achieved by means appropriate to the labelling molecule or moietyused in the assay and is typically by optical means. For example,luminescence emitted from cells or from a fluorescent labelled assayreagent may be detected in the microfabricated apparatus by the use ofimaging apparatus incorporating a CCD camera, or by the use of afluorimeter. Detection of emitted fluorescence may be achieved throughthe body of the disc (18) where the disc is wholly constructed of atransparent material or alternatively through windows of transparentmaterial which have been fabricated into the body of the disc.

[0052] As an alternative to non-radioactive detection, the apparatus ofthe present invention may be used in conjunction with radioactivedetection utilising the scintillation proximity technique. For example,scintillant-containing beads may be introduced into the apparatus.Alternatively the microfabricated apparatus of the invention may haveincorporated a scintillant containing layer into or onto an interiorsurface of the cell growth chamber (2, 7) and/or the assay chamber (3,11). The region of the apparatus containing the scintillant beads orscintillant surface is preferably optically transparent so as to enablethe material to transmit light at a given wavelength with optimumefficiency. The scintillant-containing region can be composed of anytransparent material containing scintillant, eg. a scintillant glassbased on lanthanide metal-containing compounds, or a plastic materialsuch as polystyrene or polyvinyltoluene, into which is incorporated thescintillant substance.

[0053] Suitable scintillant substances can include aromatic hydrocarbonssuch as p-terphenyl, p-quaterphenyl and their derivatives, andderivatives of oxazoles and 1,3,4-oxadiazoles, for example,2-(4-t-butylphenyl)-5-(4-biphenylyl)-1,3,4-oxadiazole and2,5-diphenyloxazole. A wavelength shifter such as1,4-bis(5-phenyl-2-oxazolyl)benzene, or 9,10-diphenylanthracene, mayalso be included.

[0054] A binding moiety such as one member of a specific binding pairmay be immobilised onto the surface of the bead or scintillant layer soas to specifically bind with an analyte which may be derived from cellsused in the assay process. Suitable specific binding pair members withwhich the invention is useful include biotin, streptavidin, protein A,antibodies, lectins, hormone receptors, nucleic acid probes, DNA bindingproteins and the like. It is to be understood that either member of thespecific binding pair may be attached and immobilised for binding to acomplementary member of the specific binding pair.

[0055] Typical radioistopes that may be used to label the assay reagentinclude those commonly used in biochemistry such as [³H], [¹²⁵I], [³⁵S]and [³³P] but does not preclude the use of other isotopes. Detectionmethodologies based on scintillation proximity are well known (see forexample U.S. Pat. No. 4,568,649, Bertoglio-Matte,J.). Detection mayutilise a number of modalities to be combined to allow all assays to bemeasured simultaneously or in rapid succession. Alternatively anappropriate imaging format may provide suitable detection means for bothradioactive and non-radioactive assays utilising the apparatus.

[0056] In some uses, some assay areas will be redundant; that is, notall elements of the apparatus will be used every time. It is envisagedthat the user will decide what type of assays it is required to performand then select appropriate control means for directing liquid flowwithin the apparatus. Consequently, such structures will be compatiblewith a range of assay procedures and protocols which require differentcomplexities of fluid movement. For example, detection of a GFP linkedreporter gene will require a simple well structure to allow measurementof fluorescence. In contrast, measurement of an analyte secreted fromcells, for example the measurement of a cytokine by immunoassay, ormeasurement of cellular mRNA following lysis, will require a morecomplicated structure to separate the cells from the secreted analyteprior to the analysis of that analyte

[0057] The invention is further illustrated by reference to thefollowing examples.

EXAMPLES 1

[0058] A stock culture of HeLa cells was grown in Dulbecco's ModifiedEagles Medium (DMEM) supplemented with 10% calf serum and L-glutamine ina plastic flask using standard tissue culture conditions. Cells wereharvested by trypsinisation and the resulting suspension concentrated bycentrifugation to give a cell concentration of 10⁷ cells/ml. Aliquots ofthe cell suspension (1 μl) were applied to openings in the surface of amicrofabricated structure of the type shown in FIG. 1, produced byinjection moulding, with internal channels of depth 50 μm and width 100μm.

[0059] The cells suspension was moved along the inlet channels withinthe disc to circular cell growth chambers of depth 50 μm and diameter500 μm and the cells allowed to attach and grow. Following incubationfor 48 hours in a tissue culture incubator (37° C./95% RH) the cellswere examined for cell density, morphology and viability. Visibleexaminations by phase contrast microscopy showed cell populationsgrowing in microstructures to have the same density and morphology ascontrol cultures grown from the same parent stock and maintained instandard tissue culture plasticware. Cells were subsequently tested forviability using a commercial test kit (LIVE/DEAD Viability Kit,Molecular Probes, Oregon, L-3224) by flushing growth medium from thecells within the device, washing the cells with a phosphate bufferedsaline solution (PBS) and introducing a solution of the fluorescentassay reagents into the cell growth chambers. Subsequent examination byfluorescent microscopy showed that cells growing in microstructures hadmaintained a cell viability of >95%, comparable with control cells grownunder standard tissue culture conditions.

EXAMPLE 2

[0060] A microfabricated plastic disc of the type shown in FIG. 1,produced by injection moulding which contained a number of radialinternal channels of depth 50 μm and width 100 μm was selectivelysurface modified by exposure to a 500W UV lamp at a distance of 20 cmfor 30 minutes through a metal mask such that UV light impinged on thesurface only on regions defined by the mask.

[0061] A stock culture of HeLa cells was grown in Dulbecco's ModifiedEagles Medium (DMEM) supplemented with 10% calf serum and L-glutamine ina plastic flask using standard tissue culture conditions. Cells wereharvested by trypsinisation and the resulting suspension concentrated bycentrifugation to give a cell concentration of 10⁷ cells/ml. Aliquots ofthe cell suspension (1 μl) were applied to openings in the surface ofthe device and cells moved along the channels by rotation about the discaxis (1000 rpm for 30 seconds). Following incubation for 18 hours in atissue culture incubator (37° C./95% RH) cells were examined by phasecontrast microscopy. Cells were found to be preferentially growing inregions of the disc previously exposed to UV light, cells in otherregions were observed to be sparser and less well attached. The disc wasthen rotated at 1000 rpm for 30 seconds and cells re-examined. Followingspinning cells in UV exposed areas were observed to have remainedattached to the plastic surface and remained morphologically identicalto control cells; in contrast cells on non-exposed surfaces were totallyremoved by the centrifugal force generated by the spinning disc.

EXAMPLE 3

[0062] HeLa cells grown and harvested as described above were introducedinto the device of Example 2 into micro channels which contained mouldedpillars of various dimensions dispersed in the channels so as to act asbarriers to cell movement in the channels. A suspension of cell wasintroduced into the channels and the structure incubated for 18 hours toallow the cells to settle and grow. Subsequent examination revealed thatin channels where the gaps between pillars were 10×60 μm or greatercells were observed to have moved freely past the barriers. In contrastwhere gaps between pillars were 10×20 μm or less, cells were preventedfrom passing the barrier when transported by liquid flow in suspension.However on subsequent incubation and growth cells were observed tomigrate past such barriers by slow deformation. Such barrier structuresmay prove useful for study of cell movement or migration in response tochemical or physical stimuli.

EXAMPLE 4

[0063] HeLa cells transfected to yield stable expression of aCalreticulin-GFP fusion protein were introduced into a moulded apparatusof the type shown in FIG. 1, supporting channels of 100 μm width and 100μm depth connected to circular chambers of 600 μm and 1000 μm diameterand 100 μm depth. Cells were incubated for 18 hours in a tissue cultureincubator and examined by confocal fluorescence microscopy. Cells grownin microfabricated structures showed the same level of GFP expression ascontrol cells grown under conventional tissue culture conditions.

EXAMPLE 5

[0064] HeLa cells were grown and introduced into a microfabricated discas described in Example 1. Following incubation for 18 hours growthmedium was removed from cells and replaced with media containingdifferent concentrations of the membrane permeabilising agent digitoninin the range 0-0.2 mg/ml (w/v) and cells incubated in the presence ofdigitonin for 10 minutes. Thereafter the viability of the cells wasmeasured as described in Example 1. Results were found to be equivalentto cells exposed to the same dose range of digitonin in a microtitreplate based assay.

1. Apparatus microfabricated for performing cell growth and cell basedassays in a liquid medium, said apparatus comprising: a) a base platesupporting a plurality of micro-channel elements, each comprising a cellgrowth chamber, an inlet channel for supplying liquid sample thereto andan outlet channel for removal of liquid sample therefrom; b) a coverplate positioned over said base plate said cover plate extending oversaid elements so as to define said chambers and connecting channels;said cover plate being supplied with holes to provide access to saidchannels; and c) means, incorporated in said cell growth chambers, forcell attachment and cell growth.
 2. Apparatus according to claim 1wherein said base plate comprises a rotatable disc which ismicrofabricated to provide a sample introduction port located towardsthe centre of the disc and connected to an annular sample reservoir, andwherein said micro-channel elements are radially dispersed on said discwith their respective input channels connected to receive sample fromsaid reservoir.
 3. Apparatus according to claim 1 or claim 2 whereinsaid cover plate is fabricated from a gas permeable plastics material.4. Apparatus according to any one of claims 1 to 3 wherein said meansfor cell attachment and cell growth comprises selectively treating ormodifying at least a portion of a surface of said cell growth chamber.5. Apparatus according to any one of claims 1 to 3 wherein means forcell attachment and cell growth comprises one or more microcarrier beadslocated in said cell growth chamber, wherein each of said microcarrierbeads provides for cell attachment and cell growth.
 6. Apparatusaccording to any one of claims 1 to 5 wherein the cell growth chamberfurther includes raised moulded features disposed on the base portion ofthe cell growth chamber to form pillars.
 7. Apparatus according to anyone of claims 1 to 6 wherein the cross-sectional area of said inletchannel is greater than that of said outlet channel.
 8. Apparatusaccording to claim 7 wherein the cross-sectional area of said outletchannel is between 0.99 and 0.01 times that of said inlet channel. 9.Apparatus according to any one of claims 1 to 8 wherein at least some ofsaid micro-channel elements each further comprises one or more assaychambers for performing assays involving cellular constituents andconnected in line between said cell growth chamber and said outletchannel.
 10. Apparatus according to claim 9 wherein the assay chamber orchambers are connected to each other and to said cell growth chamber byan intermediate channel or channels in the order of: inlet channel, cellgrowth chamber, intermediate channel 1, assay chamber 1, intermediatechannel 2 (if present), assay chamber 2 (if present) . . . , outletchannel, and wherein the cross-sectional areas of the respectivechannels reduce progressively from the inlet channel to the outletchannel.
 11. Apparatus according to claim 10 wherein the cross-sectionalarea of the or each intermediate channel and the outlet channel isbetween 0.99 and 0.01 times that of the immediately preceding (upstream)channel.
 12. Apparatus according to any one of claims 1 to 11 whereinthere is provided in or on an interior surface of one or more of saidchambers a layer comprising a scintillant substance.
 13. Apparatusaccording to claim 12 wherein the layer comprising a scintillantsubstance includes a binding moiety bound thereto, said binding moietybeing a member of a specific binding pair selected from biotin,streptavidin, protein A, antibodies, lectins, hormone-receptors, nucleicacid probes, and DNA-binding proteins.
 14. A method for determining theeffect of a test substance on a cellular activity or physical parameterby the use of an apparatus as defined in claims 1-13, which methodcomprises: a) providing a suspension of cells in a fluid medium; b)introducing said cells into said apparatus and causing said cells to betransported to one or more cell growth chambers in said apparatus; c)providing one or more samples of test substances whose effect upon thecells is to be measured under conditions so as to cause said cells to beexposed to said substances; d) determining the effect of the testsubstances on said cells by means of optical detection.
 15. A methodaccording to claim 14 wherein cells are cultured adhering to a surfacewithin the apparatus prior to the introduction of the test substances.16. A method according to claims 14 or 15 wherein there are providedfollowing step c) one or more assay reagents and dispersing saidreagents to one or more reaction chambers in said apparatus.
 17. Amethod according to claim 16 wherein at least one of said assay reagentsis labelled with a detectable label selected from fluorescent labels,chemiluminescent labels, bioluminescent labels, enzyme labels andradioactive labels.
 18. A method for measuring a cellular analyte by theuse of the apparatus as defined in claims 1-13, which method comprises:a) providing a suspension of cells containing an analyte to be measuredin a fluid medium; b) introducing said cells into said apparatus andcausing the cells to be transported to one or more cell growth chambersin the apparatus; c) allowing cells to grow; d) providing one or moreassay reagents and dispersing said reagents to one or more chambers insaid apparatus; e) measuring the cellular analyte by optical means. 19.A method according to claim 18 wherein at least one of said assayreagents is labelled with a detectable label selected from fluorescentlabels, chemiluminescent labels, bioluminescent labels, enzyme labelsand radioactive labels.